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W C Greene

Publications and source records attributed to W C Greene.

At least 181 records · Page 10Linked to original sources
PubMed1985

Isolation and expression of complementary DNAs encoding the human interleukin 2 receptor.

Complementary DNAs corresponding to the human receptor for interleukin 2 (IL-2) have been molecularly cloned, sequenced, and expressed in COS-1 cells. The human genome appears to contain a single structural gene for this receptor; however, when transcribed at least two messenger RNAs (mRNAs) are produced which vary in length due to the use of different polyadenylation signals. Sequence analysis of the cloned complementary DNAs indicates an alternate pathway of mRNA processing for this receptor. Splicing of a 216 base pairs segment contained within the protein coding region results in an mRNA unable to code for the IL-2 receptor. In contact complementary DNAs corresponding to unspliced mRNA encode membrane receptors which bind both IL-2 and anti-Tac (monoclonal anti-IL-2 receptor antibody). Analysis of the deduced amino acid sequence reveals that the receptor is composed of 272 amino acids including a signal peptide 21 amino acids in length. Hydrophobicity analysis suggests a single 19 amino acid transmembrane domain. A short intracytoplasmic domain composed of 13 amino acids is present at the carboxy terminus and contains three potential phosphate acceptor sites (serine and threonine but not tyrosine) and typical positively charged amino acids presumably involved in cytoplasmic anchoring. Two sites for N-linked glycosylation sites and numerous extracytoplasmic O-linked glycosylation sites are present.

Amino Acid Sequence↗
PubMed1985

Effect of wheat germ agglutinin on the interleukin pathway of human T lymphocyte activation.

Wheat germ agglutinin (WGA) inhibits proliferation of human peripheral blood mononuclear cells (PBMC) induced by mitogens and antigens. We investigated the mechanism by which WGA inhibits PHA-induced human lymphocyte proliferation with regard to the interleukin pathway. Our data revealed that although PBMC-proliferation was markedly suppressed by WGA, levels of IL 2 activity in WGA-inhibited cultures were not reduced, but instead were increased, suggesting failure to utilize IL 2. Furthermore, the addition of exogenous IL 2 failed to overcome the suppression. Consistent with these observations, culturing PBMC with PHA plus WGA markedly decreased the number of high-affinity IL 2 receptor per cell, as determined by binding of purified [3H]IL 2, relative to cultures containing PHA alone. WGA immobilized on support beads bound detergent-solubilized IL 2 receptors from PHA-activated T cells, but did not bind human IL 2. However, WGA did not competitively block the binding of [3H]IL 2 to PHA-induced lymphoblasts. These results suggest that WGA inhibits lymphocyte proliferation by binding to and decreasing the number of high-affinity IL 2 receptors displayed on T cells, without impairing IL 2 production.

Cells, Cultured↗
PubMed1985

Nucleotide sequence and expression of a mouse interleukin 2 receptor cDNA.

We have isolated and sequenced a 1.3 kilobase (kb) cDNA that contains the entire 804 base pair coding region of the murine interleukin 2 (IL 2) receptor. A comparison with the human IL 2 receptor sequence revealed regions of high conservation, including the short (11 amino acid) intracytoplasmic domain, as well as an internally repeated sequence that is consistent with the receptor containing at least two distinct extracytoplasmic domains. When inserted into the expression vector pcEXV-3, this cDNA directs functional membrane expression of the IL 2 receptor on transfected COS 7 cells. An analysis of cytoplasmic RNA from IL 2 receptor-bearing T cells reveals five distinct polyadenylated transcripts ranging in size from 1.7 to 4.7 kb. All five transcripts are evident in both concanavalin A-stimulated splenic T cells and in the IL 2-dependent line CTLL. Some of this size heterogeneity is due to different sites of polyadenylation, resulting in mRNAs that differ in the 3' untranslated region. We cannot exclude the possibility of alternative splicing, however, which may result in distinct forms of the IL 2 receptor on the cell surface.

Animals↗
PubMed1985

Monoclonal antibody OKT11A inhibits and recombinant interleukin 2 (IL 2) augments expression of IL 2 receptors at a pretranslational level.

The expression of receptors for interleukin 2 (IL 2) represents a critical event regulating the growth of normal T lymphocytes. We investigated the effects of the inhibitory monoclonal antibody OKT11A (anti-sheep erythrocyte receptor) and of purified recombinant IL 2 (rIL 2) on the expression of IL 2 receptors by activated T cells at both the protein and the mRNA levels. Adding OKT11A antibody (0.5 microgram/ml) to phytohemagglutinin (PHA)-stimulated cultures of human peripheral blood mononuclear cells (PBMC) markedly suppressed cellular proliferation (assessed by [3H]thymidine incorporation) and IL 2 receptor expression (determined by immunofluorescence assay by using the anti-IL 2-receptor antibody, anti-Tac). Northern blot analysis performed with the use of a cDNA probe specific for the human IL 2 receptor gene demonstrated that OKT11A antibody also decreased the accumulation of IL 2 receptor mRNA induced by PHA in PBMC. Purified rIL 2 (10 U/ml) alone had little effect on the expression of IL 2 receptors in unstimulated PBMC cultures. In combination with PHA or with PHA plus OKT11A, however, rIL 2 augmented both the expression of IL 2 receptor protein on PBMC and the accumulation of IL 2 receptor mRNA in PBMC. Adding anti-Tac antibody to PBMC cultures to block the interaction of IL 2 with its receptor diminished the accumulation of IL 2 receptor mRNA induced by PHA. Taken together, these data demonstrate that OKT11A antibody inhibits and IL 2 augments expression of IL 2 receptors on PHA-stimulated T cells, at least in part, at a pretranslational level.

Adjuvants, Immunologic↗
PubMed1984

Expression of interleukin 2 receptors on activated human B cells.

Using anti-Tac, a monoclonal anti-interleukin 2 (IL-2) receptor antibody, we have explored the possibility that certain activated B cells display receptors for IL-2. Resting normal B cells and unselected B cell lines established from normal individuals were Tac antigen negative. In contrast, the cell surface Tac antigen expression was demonstrable on 6 of 10 B cell lines from patients with Burkitt's lymphoma, 5 of 6 B cell lines derived from patients with HTLV-I-associated adult T cell leukemia (including all four that had integrated HTLV-I into their genome), and on certain normal B cells activated with pokeweed mitogen. Furthermore, cloned Epstein-Barr virus-transformed B cell lines derived from Tac-positive normal B cells continued to express the Tac antigen in long-term cultures and manifested high affinity IL-2 receptors identified in binding studies with purified radiolabeled IL-2. The line 5B4 developed in the present study could be induced with purified JURKAT-derived or recombinant IL-2 to express a larger number of IL-2 receptors. Furthermore, the addition of IL-2 to the 5B4 B cell line augmented IgM synthesis, which could be blocked by the addition of anti-Tac. The size of the IL-2 receptors expressed on the cloned normal B cell lines was similar (53,000-57,000 daltons) to that of receptors on phytohemagglutinin-stimulated T cell lymphoblasts. Thus, certain malignant and activated normal B cells display the Tac antigen and manifest high affinity receptors for IL-2. These data suggest that IL-2 may play a role in the differentiation of activated B cells into immunoglobulin-synthesizing and -secreting cells.

Antibodies, Monoclonal↗
PubMed1984

Low and high affinity cellular receptors for interleukin 2. Implications for the level of Tac antigen.

Interleukin 2 promotes proliferation of T cells by virtue of its interaction with a high-affinity cell surface receptor. This receptor is a 55,000 mol wt glycoprotein that is also recognized by the murine monoclonal antibody, anti-Tac. Quantitative binding studies with radiolabeled IL-2 and anti-Tac, however, initially indicated far more antibody binding sites per cell than IL-2 binding sites. Extension of the IL-2 binding analysis to concentrations several thousand-fold higher than that necessary for the T cell proliferative response demonstrated the existence of a class (or classes) of low-affinity IL-2 binding sites. Inclusion of the low-affinity IL-2 binding greatly reduced the quantitative discrepancy in the ligand binding assays. That the low-affinity binding, as well as the high-affinity interaction, was associated with the Tac molecule was indicated by the finding that the antibody could substantially or totally block the entire spectrum of IL-2 binding and by the finding that IL-2 could in turn block all radiolabeled anti-Tac binding. The low-affinity sites were found on activated T cells, several human and murine T cell lines and two examples of Tac-positive B cells. The physiological role of the low-affinity IL-2 binding sites and the molecular changes in the Tac protein that give rise to the affinity differences remain open to investigation.

Animals↗
PubMed1984

Cyclosporin A inhibits T-cell growth factor gene expression at the level of mRNA transcription.

Cyclosporin A (CsA) is a potent immunosuppressive agent, now gaining wide application in human organ transplantation. The immunosuppressive activity of CsA is at least in part due to inhibition of lymphokine production by activated T lymphocytes. Specifically, inhibition of T-cell growth factor (TCGF; also designated interleukin 2) production appears to be an important pathway by which CsA impairs T-cell function. To define further both the specificity of CsA and the level at which it interferes with lymphokine gene expression, we have studied its effects on TCGF mRNA accumulation as well as TCGF gene transcription. These studies were performed with a cloned human leukemic T-cell line (Jurkat, subclone 32), which can be induced with phytohemagglutinin and phorbol 12-myristate 13-acetate to produce large amounts of TCGF. In these cells, high levels of TCGF mRNA were present in induced but not in uninduced Jurkat cells as judged by hybridization to a cloned human TCGF cDNA probe. CsA completely inhibited induced TCGF mRNA accumulation at concentrations of 0.3-1.0 microgram/ml, whereas low levels of appropriately sized TCGF mRNA were present at 0.01 microgram/ml. In nuclear transcription experiments, CsA inhibited the synthesis of TCGF transcripts in a dose-dependent manner with complete inhibition at a concentration of 1 microgram/ml. In contrast, CsA did not inhibit the expression of two other inducible genes, TCGF receptor and HT-3. Further, HLA gene expression was also less affected than TCGF in CsA-treated cells. These data suggest a relatively selective action of CsA on TCGF gene transcription.

Cell Line↗
PubMed1984

Functional and phenotypic comparison of human T cell leukemia/lymphoma virus positive adult T cell leukemia with human T cell leukemia/lymphoma virus negative Sézary leukemia, and their distinction using anti-Tac. Monoclonal antibody identifying the human receptor for T cell growth factor.

Adult T cell leukemia (ATL) and Sézary leukemia are malignant proliferations of T lymphocytes that share similar cell morphology and clinical features. ATL is associated with HTLV (human T cell leukemia/lymphoma virus), a unique human type C retrovirus, whereas most patients with the Sézary syndrome do not have antibodies to this virus. Leukemic cells of both groups were of the T3, T4-positive, T8-negative phenotype. Despite the similar phenotype, HTLV-negative Sézary leukemic cells frequently functioned as helper cells, whereas some HTLV-positive ATL and HTLV-positive Sézary cells appeared to function as suppressors of immunoglobulin synthesis. One can distinguish the HTLV-positive from the HTLV-negative leukemias using a monoclonal antibody (anti-Tac) that appears to identify the human receptor for T cell growth factor (TCGF). Resting normal T cells and most HTLV-negative Sézary cells were Tac-negative, whereas all ATL cell populations were Tac-positive. The observation that ATL cells manifest TCGF receptors suggests the possibility that an abnormality of the TCGF-TCGF receptor system may partially explain the uncontrolled growth of these cells.

Adult↗
PubMed1984

Regulation of interleukin 2 receptor expression: effects of phorbol diester, phospholipase C, and reexposure to lectin or antigen.

Anti-Tac monoclonal antibody identifies the receptor for interleukin 2 (IL 2, or T cell growth factor) present on activated human T lymphocytes. By using tritiated anti-Tac, we now report a sensitive and specific binding assay to evaluate cell surface IL 2 receptor expression. IL 2 receptors on human peripheral blood lymphocytes can be detected within 6 hr after PHA stimulation. PHA-induced receptor expression is inhibited by actinomycin D and cycloheximide, but not by mitomycin C, suggesting a requirement for de novo RNA and protein synthesis, but not DNA synthesis. Scatchard analysis of [3H]-anti-Tac binding to lymphocytes stimulated with PHA for 3 days revealed from 20,000 to 60,000 molecules of antibody bound per cell, and a Kd of 1 to 3 x 10(-10) mol/l. Sequential binding studies of activated human lymphocytes maintained in long-term culture with IL 2 demonstrated a progressive decline in receptor number correlating with diminished growth rate. Restimulation with lectin or antigen increased the number of IL 2 receptors, suggesting that IL 2 dependent immune responses may be regulated, at least in part, by IL 2 receptor expression. Receptor number was also increased by PMA. Moreover, similar effects were produced by incubation with phospholipase C but not interleukin 1. Because both PMA and phospholipase C result in activation of protein kinase C, these data suggest the possibility that activation of protein kinase C may induce IL 2 receptor expression.

Antigens, Surface↗
PubMed1984

Interleukin-2 receptor expression in retrovirus associated adult T-cell leukemia.

Interleukin-2 (IL-2) is a lymphokine synthesized by some T cells following activation. Resting T cells do not express IL-2 receptors but receptors are rapidly expressed on T cells following the interaction of antigens, mitogens, or monoclonal antibodies with the antigen specific T-cell receptor complex. Using anti-Tac a monoclonal antibody that recognizes the IL-2 receptor, the receptor has been purified. The receptor is a 33 kdalton peptide that is post-translationally glycosylated to a 55 kdalton mature form. Mature receptors contain both N-linked and O-linked sugars and are both sulfated and phosphorylated. Using an oligonucleotide probe, based on the N-terminal amino acid sequence, cDNAs encoding this receptor have been cloned, sequenced and expressed. The addition of anti-Tac to in vitro culture systems blocks the IL-2 induced DNA synthesis of IL-2 dependent T-cell lines and inhibits soluble auto- and alloantigen induced T-cell proliferation. Furthermore, it prevents the generation of cytotoxic and suppressor effector T cells. The anti-receptor antibody also inhibits lectin stimulated immunoglobulin synthesis and the sequential expression of late appearing activation antigens on T cells. Normal resting T cells and most leukemic T-cell populations do not express IL-2 receptors however the leukemic cells of all patients with human T-cell leukemia/lymphoma virus (HTLV-I) associated, adult T-cell leukemia (ATL) examined expressed the Tac antigen. In HTLV-I infected cells the 42 kdalton long open reading frame (LOR) protein encoded in part, by the pX region of HTLV-I may act as a transacting transcriptional activator that induces transcription of the IL-2 receptor gene thus providing an explanation for the constant association of HTLV-I infection of lymphoid cells and IL-2 receptor expression. The constant display of large numbers of IL-2 receptors which may be aberrant in the ATL cells may play a role in the uncontrolled growth of these leukemic T cells. Patients with the Tac positive ATL are being treated with an anti-Tac monoclonal antibody directed towards this growth factor receptor.

Antibodies, Monoclonal↗
PubMed1984

Phorbol diester induces expression of Tac antigen on human acute T lymphocytic leukemic cells.

In general, the human acute T lymphocytic leukemias are composed of malignant expansions of immature T cells lacking membrane receptors for T cell growth factor (TCGF, interleukin 2) and significant immunoregulatory activity. We investigated whether cultured acute lymphocytic leukemic T cell lines can be induced to differentiate and express the Tac antigen, a cell surface protein that contains a TCGF-binding site, after exposure to phorbol 12-myristate 13-acetate (PMA) and/or phytohemagglutinin (PHA). Reactivity of anti-Tac with induced leukemic T cells was studied by three techniques, including: 1) flow microfluorometry; 2) specific binding of [3H]anti-Tac; and 3) receptor immunoprecipitation with anti-Tac and analysis by SDS-PAGE. After exposure to PMA with or without PHA, both JURKAT and HSB-2 acute lymphocytic leukemic T cells displayed Tac antigen within 6 to 8 hr. Induction of receptor expression was blocked by actinomycin D, suggesting a requirement for new mRNA transcription. Induced JURKAT cells contained approximately 7000 Tac molecules per cell, and the binding of anti-Tac to these cells was blocked in a dose-related manner by purified TCGF but not by insulin or purified recombinant interferon-alpha. SDS-PAGE analysis of anti-Tac immunoprecipitates demonstrated that receptors present on induced JURKAT cells were 2000 to 3000 daltons smaller than those present on PHA-activated normal lymphoblasts or induced HSB-2 cells. Induction of JURKAT cells with both PHA and PMA resulted in marked secretion of TCGF as well as the appearance of Tac antigen. After activation of these cells with PMA alone, Tac antigen was similarly expressed, but the level of TCGF synthesis was less than 1% of that obtained after dual induction with PHA and PMA. These data indicate that the signals required for TCGF synthesis and Tac expression are not identical, and furthermore that induction of Tac antigen and TCGF is not obligately linked in these cells.

Antigens, Surface↗
PubMed1984

Expression of receptors for interleukin 2: Role in the commitment of T lymphocytes to proliferate.

We examined the role of IL 2 and IL 2 receptors in human T lymphocyte proliferation induced by neuraminidase and galactose oxidase (NAGO)-treated autologous macrophages. T lymphocytes cultured with these aldehyde-bearing macrophages developed responsiveness to IL 2 after as few as 2 to 4 hr of activation and exhibited maximal responsiveness to IL 2 after 18 to 24 hr of activation. This early expression of IL 2 receptors was also shown by the direct binding of a monoclonal anti-IL 2 receptor antibody (anti-Tac) to the activated T lymphocytes. The production of IL 2 by T lymphocytes cultured with NAGO-treated macrophages closely paralleled the induction of IL 2 receptors on the T lymphocytes. IL 2 production began after 4 to 8 hr of activation and peaked at approximately 18 hr. Although the production of IL 2 is strictly dependent upon accessory cell function, the expression of receptors for IL 2 seems to be relatively independent of accessory cells. Despite early expression of receptors for IL 2 and early production of IL 2 by T lymphocytes during activation, T lymphocytes were not committed to proliferate in the absence of IL 2 until more than 24 hr of incubation with NAGO-treated macrophages had elapsed. The commitment to proliferate increased after 24 hr of activation until, after more than 40 hr of activation, the cells proliferated equally well in the presence or absence of IL 2. Proliferation of uncommitted, IL 2 receptor-bearing T lymphocytes was inhibited by interfering with IL 2 binding to its receptor by IL 2 receptor blockade with the anti-Tac antibody. In contrast, proliferation of T lymphocytes committed to proliferate was not affected by IL 2 receptor blockade with the anti-Tac antibody. Taken together, these data suggest three phases of T lymphocyte activation. The first phase requires mitogen (or antigen) to induce expression of IL 2 receptors and production of IL 2 by the T lymphocytes. Accessory cells are strictly required for IL 2 production during this activation phase, but they may not be necessary for expression of IL 2 receptors. The second phase is an IL 2-dependent phase that requires the interaction of IL 2 with the newly expressed IL 2 receptors. The third phase is a commitment of the activated T lymphocytes to proliferate that is independent of both mitogen and IL 2.

Adult↗
PubMed1983

Direct demonstration of the identity of T cell growth factor binding protein and the Tac antigen.

Radiolabeled molecules from detergent-solubilized human T cell blasts were fractionated on affinity supports coupled with T cell growth factor (TCGF) and anti-Tac antibody. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis indicated that a glycoprotein of approximately 58,000 mol wt was bound in both cases. Sequential binding to the two affinity supports demonstrated that the molecules recognized in each instance were identical. Thus, the Tac antigen contains the cellular binding site for TCGF. Note added in proof: Preliminary binding experiments using high concentrations of [3H]leucine, lysine TCGF with a low specific radioactivity indicate the existence of a sizeable pool of receptor sites with an affinity 2,000-10,000 times lower than that of the high affinity receptors measured in Fig. 1. Such sites may explain the numerical discrepancy between early TCGF binding experiments (5) and the binding of the anti-Tac antibody. The hypothesis that the anti-Tac antibody apparently reacts with both classes of receptor would explain its effect on the physiological response (high affinity binding) and the high level of Tac antigen on the cell surface.

Antibodies, Monoclonal↗
PubMed1983

Polymorphonuclear neutrophils express the common acute lymphoblastic leukemia antigen.

Monoclonal antibodies J5, VIL-A1, and BA-3, known to react with the common acute lymphoblastic leukemia antigen (CALLA) were found to specifically stain normal human polymorphonuclear neutrophils (PMN). The antigen detected on PMN had a molecular weight (95,000-110,000 mol wt) close to that of CALLA (95,000-100,000 mol wt) and thus these surface membrane antigens are likely related, if not identical. The fluorescent staining intensity of PMN is comparable to that of CALLA-positive leukemic cells and the presence of PMN in patient samples could potentially produce false-positive results in diagnosis.

Adult↗
PubMed1983

Evidence for an interleukin-independent pathway for human lymphocyte activation.

Though lectin mitogen stimulation of T-cell proliferation is an interleukin 1- (IL 1), interleukin 2- (IL 2) dependent process, the calcium ionophore A23187 may be able to initiate T-lymphocyte proliferation by an additional pathway. That the action of A23187 is IL 1 independent was demonstrated by its ability to stimulate monocyte-depleted cells without the addition of exogenous IL 1. The IL 2 independence of A23187 was indicated by (i) the inability of exogenous IL 2 to augment A23187-induced proliferation and (ii) the inability of the monoclonal antibody anti-Tac (with specificity for the human IL 2 receptor) to inhibit proliferation mediated by A23187. By contrast, in cultures stimulated with the lectin mitogen phytohemagglutinin, additional IL 2 considerably increased proliferation, whereas anti-Tac routinely caused 60-90% inhibition. Although the ionophore caused some IL 2 production and resulted in IL 2 receptor expression, quantitative studies showed that our results could not be explained by excessive amounts of endogenous IL 2 interfering with the blocking action of the antibody. Therefore, these data suggest that the action of A23187 as a human lymphocyte mitogen may be the result of at least two pathways--one dependent on the interleukin-cell interactions and the other independent of these mediators.

Anti-Bacterial Agents↗